GPR114/ADGRG5 is activated by its tethered peptide agonist because it is a cleaved adhesion GPCR.

Family B2 or adhesion G protein-coupled receptors (AGPCRs) are distinguished by variable extracellular regions that contain a modular protease, termed the GPCR autoproteolysis-inducing domain that self-cleaves the receptor into an N-terminal fragment (NTF) and a C-terminal fragment (CTF), or seven transmembrane domain (7TM). The NTF and CTF remain bound after cleavage through noncovalent interactions. NTF binding to a ligand(s) presented by nearby cells, or the extracellular matrix anchors the NTF, such that cell movement generates force to induce NTF/CTF dissociation and expose the AGPCR tethered peptide agonist. The released tethered agonist (TA) binds rapidly to the 7TM orthosteric site to activate signaling. The orphan AGPCR, GPR114 was reported to be uncleaved, yet paradoxically capable of activation by its TA. GPR114 has an identical cleavage site and TA to efficiently cleave GPR56. Here, we used immunoblotting and biochemical assays to demonstrate that GPR114 is a cleaved receptor, and the self-cleavage is required for GPR114 TA-activation of Gs and no other classes of G proteins. Mutagenesis studies defined features of the GPR114 and GPR56 GAINA subdomains that influenced self-cleavage efficiency. Thrombin treatment of protease-activated receptor 1 leader/AGPCR fusion proteins demonstrated that acute decryption of the GPR114/56 TAs activated signaling. GPR114 was found to be expressed in an eosinophilic-like cancer cell line (EoL-1 cells) and endogenous GPR114 was efficiently self-cleaved. Application of GPR114 TA peptidomimetics to EoL-1 cells stimulated cAMP production. Our findings may aid future delineation of GPR114 function in eosinophil cAMP signaling related to migration, chemotaxis, or degranulation.

Hexahydroquinoline Derivatives Are Selective Agonists for the Adhesion G Protein-Coupled Receptor ADGRG1/GPR56.

GPR56 is a widely expressed adhesion GPCR (AGPCR) that has pleotropic roles in brain development, platelet function, cancer, and more. Nearly all AGPCRs possess extracellular regions that bind protein ligands and conceal a cryptic tethered peptide agonist. AGPCR reception of mechanical or shear force is thought to release the tethered agonist permitting its binding to the AGPCR orthosteric site for consequent activation of G protein signaling. This multistep mechanism of AGPCR activation is difficult to target, emphasizing the need for tool compounds and potential therapeutics that modulate AGPCRs directly. We expanded our cell-based pilot screen for GPR56 small molecule activators to screen >200,000 compounds and identified two promising agonists: 2-(furan-2-yl)-1-[(4-phenylphenyl)carbonyl]pyrrolidine, or compound 4, and propan-2-yl-4-(2-bromophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate, or compound 36. Both compounds activated GPR56 receptors enginered to have impaired tethered agonists and/or be cleavage deficient. Compound 4 activated a subset of group VIII AGPCRs while compound 36 had exclusive specificity for GPR56 among the GPCRs tested. Compound 36 SAR analysis identified an analog with the isopropyl R group replaced with a cyclopentyl ring and the electrophilic bromine replaced with a CF3 group. Analog 36.40 had 40% increased potency over compound 36 and was 20-fold more potent than synthetic peptidomimetics designed from the GPR56 tethered agonist. The new GPCR56 tool compounds discovered in this screen may be used to further advance understanding of GPR56 function and aid development of AGPCR-targeted therapeutics. SIGNIFICANCE STATEMENT: Adhesion G protein coupled receptors (AGPCRs) are a large, clinically relevant class of GPCRs with no available therapeutics, in part due to their unique mechanism of activation. GPR56 is a widely expressed model AGPCR involved in cancer metastasis, hemostasis, and neuron myelination. In the present study, we identified novel small molecule agonists for GPR56. These molecules are among the most potent identified thus far and may become useful leads in the development of a GPR56-targeted therapeutic.

The tethered peptide activation mechanism of adhesion GPCRs.

Adhesion G-protein-coupled receptors (aGPCRs) are characterized by the presence of auto-proteolysing extracellular regions that are involved in cell-cell and cell-extracellular matrix interactions1. Self cleavage within the aGPCR auto-proteolysis-inducing (GAIN) domain produces two protomers-N-terminal and C-terminal fragments-that remain non-covalently attached after receptors reach the cell surface1. Upon dissociation of the N-terminal fragment, the C-terminus of the GAIN domain acts as a tethered agonist (TA) peptide to activate the seven-transmembrane domain with a mechanism that has been poorly understood2-5. Here we provide cryo-electron microscopy snapshots of two distinct members of the aGPCR family, GPR56 (also known as ADGRG1) and latrophilin 3 (LPHN3 (also known as ADGRL3)). Low-resolution maps of the receptors in their N-terminal fragment-bound state indicate that the GAIN domain projects flexibly towards the extracellular space, keeping the encrypted TA peptide away from the seven-transmembrane domain. High-resolution structures of GPR56 and LPHN3 in their active, G-protein-coupled states, reveal that after dissociation of the extracellular region, the decrypted TA peptides engage the seven-transmembrane domain core with a notable conservation of interactions that also involve extracellular loop 2. TA binding stabilizes breaks in the middle of transmembrane helices 6 and 7 that facilitate aGPCR coupling and activation of heterotrimeric G proteins. Collectively, these results enable us to propose a general model for aGPCR activation.

GPR56/ADGRG1 is a platelet collagen-responsive GPCR and hemostatic sensor of shear force.

Circulating platelets roll along exposed collagen at vessel injury sites and respond with filipodia protrusion, shape change, and surface area expansion to facilitate platelet adhesion and plug formation. Various glycoproteins were considered to be both collagen responders and mediators of platelet adhesion, yet the signaling kinetics emanating from these receptors do not fully account for the rapid platelet cytoskeletal changes that occur in blood flow. We found the free N-terminal fragment of the adhesion G protein-coupled receptor (GPCR) GPR56 in human plasma and report that GPR56 is the platelet receptor that transduces signals from collagen and blood flow-induced shear force to activate G protein 13 signaling for platelet shape change. Gpr56-/- mice have prolonged bleeding, defective platelet plug formation, and delayed thrombotic occlusion. Human and mouse blood perfusion studies demonstrated GPR56 and shear-force dependence of platelet adhesion to immobilized collagen. Our work places GPR56 as an initial collagen responder and shear-force transducer that is essential for platelet shape change during hemostasis.

Mechanisms of adhesion G protein-coupled receptor activation.

Adhesion G protein-coupled receptors (AGPCRs) are a thirty-three-member subfamily of Class B GPCRs that control a wide array of physiological processes and are implicated in disease. AGPCRs uniquely contain large, self-proteolyzing extracellular regions that range from hundreds to thousands of residues in length. AGPCR autoproteolysis occurs within the extracellular GPCR autoproteolysis-inducing (GAIN) domain that is proximal to the N terminus of the G protein-coupling seven-transmembrane-spanning bundle. GAIN domain-mediated self-cleavage is constitutive and produces two-fragment holoreceptors that remain bound at the cell surface. It has been of recent interest to understand how AGPCRs are activated in relation to their two-fragment topologies. Dissociation of the AGPCR fragments stimulates G protein signaling through the action of the tethered-peptide agonist stalk that is occluded within the GAIN domain in the holoreceptor form. AGPCRs can also signal independently of fragment dissociation, and a few receptors possess GAIN domains incapable of self-proteolysis. This has resulted in complex theories as to how these receptors are activated in vivo, complicating pharmacological advances. Currently, there is no existing structure of an activated AGPCR to support any of the theories. Further confounding AGPCR research is that many of the receptors remain orphans and lack identified activating ligands. In this review, we provide a detailed layout of the current theorized modes of AGPCR activation with discussion of potential parallels to mechanisms used by other GPCR classes. We provide a classification means for the ligands that have been identified and discuss how these ligands may activate AGPCRs in physiological contexts.